An estimated 1.5 million people die each year from invasive fungal infections, and many millions more are afflicted by debilitating mucosal and subcutaneous mycoses. Current antifungal therapies have serious deficiencies including poor efficacy, limited spectrum of activity, patient toxicity and the emergence of resistant fungi. Consequently, mortality rates have remained disturbingly high. A major obstacle to developing effective new antifungals is the fundamental similarity of these eukaryotic pathogens and their mammalian host at the cellular level. This presents a challenge in devising therapeutic agents with pathogen selective toxicity. The objective of this proposal is to substantiate the antifungal efficacy of chemotherapeutics that target the fungal vacuole. The absence of a closely related organelle in mammalian cells suggests that the vacuole may provide an invaluable opportunity to selectively target infectious fungi. Our molecular studies have shown that disrupting vacuolar integrity in the prevalent human pathogen, Candida albicans, severely impairs its ability to colonize mammalian tissue or cause lethal infection in mice. Vacuolar function is also essential for Cryptococcus neoformans to survive within the mammalian host and cause meningoencephalitis. In either fungus, loss of vacuolar function causes a multitude of pathogenesis related phenotypes, including hypersensitivity to a variety of stresses and severely diminished expression of virulence attributes. Therefore, we hypothesize that the fungal vacuole can be exploited to develop effective new antifungal therapies because it is essential for fungal pathogenesis, and has diverged significantly from the mammalian lysosome. To test this we have devised a high-throughput screening assay that has so far identified 82 potential Vacuole Disrupting chemical Agents (VDAs). In Aim 1 of this study we will characterize the activity of these VDAs upon the fungal vacuole as well as the equivalent mammalian lysosome, and select those with potent and fungal- selective activity. In Aim 2, we will select VDAs with the greatest in vitro antifungal activity. Finally, in Aim 3 we will identify the moleculr targets or pathways upon which the most efficacious VDAs act, and use a mouse model of disseminated candidiasis to test if the inhibition of these targets is sufficient to cure an established in vivo infection. Completion of these studies will uncover the true potential of targeting the fungal vacuole as a strategy to cure life-threatening fungal infections, establish a pipeline of 'lead' compounds that can form the basis of such interventions, as well as identify and validate chemically tractable targets.